title: A refined cytochrome P540 IC₅₀ shift assay for reliably identifying CYP3A time-dependent inhibitors.
abstract: A refined cytochrome P450 (P450) enzyme IC₅₀ shift assay for more accurately screening CYP3A time-dependent inhibitors (TDIs) is presented. In contrast to the regular IC₅₀ shift assay, in which only one pair of P450 inhibition curves is generated, this modified method generates two pairs of inhibition curves; one pair of curves is created from human liver microsomal incubations with the test article in the presence or absence of NADPH (curves 1 and 2) (same as the traditional assay), and the other pair is created from new microsomal incubations with extract (compound/metabolites) of previous incubations (curves 3 and 4). To assess the true CYP3A time-dependent inhibition, we propose a new parameter, the vertical IC₅₀ curve shift (VICS), represented by vertical shift difference between the two sets of curves divided by inhibitor concentration at which maximal vertical shift of curves 1 and 2 is observed. A shift in the curves 1 and 2 could mean a time-dependent inhibition or formation of a more active inhibitory metabolite(s). The new method provides more reliable characterization of the shift as a result of a true TDI- or metabolite-mediated reversible inhibition. Nine known TDI drugs were evaluated using this refined shift assay. The derived VICS values correlated well with the reported k(inact)/K(I) values derived via the conventional dilution assay method. Thus, the refined assay can be used to identify a true TDI and quantitatively assess the inactivation potential of TDIs in a high-throughput fashion. This assay can be invaluable to screen for true P450 TDIs in the early drug discovery.
authors.affiliation: Biogen Idec, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA. Ping.li@biogenidec.com
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Chuang
authors.initials: C
authors.lastname: Lu
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Balani
authors.forename: Liang-Shang
authors.initials: LS
authors.lastname: Gan
mesh_terms: D051544:Cytochrome P-450 CYP3A; D065692:Cytochrome P-450 CYP3A Inhibitors; D055808:Drug Discovery / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000009:adverse effects* / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D013997:Time Factors
chemical_list: D065692:Cytochrome P-450 CYP3A Inhibitors; D004791:Enzyme Inhibitors; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A